The long-term goal of this project is to obtain a greater mechanistic understanding of molecular transport through the nuclear pore complex (NPC). The fulfillment of this goal is fundamentally and broadly significant in medicine, biology, chemistry, and nanoscience, and, subsequently, it will make a powerful, sustained impact on better human health. In this work, we propose and test the novel hypothesis that molecular transport through the NPC is spatially regulated at the nanometer scale. Specifically, we hypothesize that the interior of the NPC nanopore is concentrically divided into central and peripheral routes by the hydrophobic transport barriers that are rich in phenylalanine and glycine. In addition, we hypothesize that the peripheral route is more loosely blocked by more flexible barriers to be more readily permeabilized for the nuclear import of macromolecules. Significantly, this hypothesis implies that the peripheral route should be targeted for the nuclear import of therapeutic macromolecules and nanomaterials for gene therapy and nanomedicine. We will test these hypotheses by measuring the permeability of central and peripheral routes to various probe molecules by using newly developed methodologies. Our transport study will reveal not only the higher permeability of the peripheral route but also the types of the interactions, e.g., hydrophobic, steric, and electrostatic, that each transport barrier exerts on a probe molecule. Innovatively, we will directly resolve molecular transport through each route of the single NPC by using scanning electrochemical microscopy (SECM) with an unprecedentedly high spatial resolution of <10 nm. The results of this single-NPC imaging will be quantitatively compared with those of the well-established micrometer-scale SECM study of multiple NPCs. These SECM studies are focused on the passive transport of small probe molecules and they are complemented by the confocal fluorescence microscopic study of macromolecular transport through multiple NPCs. Molecular transport through multiple NPCs can be studied with pathway selectivity by employing the proteins or small molecules that can selectively block or permeabilize a target route, respectively.